Flow cytometry provides a method of analyzing and differentiating particles applicable to various clinical and research applications. Generally, flow cytometer systems irradiate particles and then sense the radiation emitted from the particle in order to identify particular physical attribute(s) of the individual particle(s) being studied. In particle sorting applications, each particle can be separated from the main population based upon the physical attributes identified as disclosed, for example, by U.S. Pat. Nos. 5,643,796; 5,602,349; and 5,602,039; and International Patent Application No. PCT/US95/13308, each hereby incorporated by reference.
During operation of flow cytometers, hydrodynamic focusing entrains particles in a fluid stream so that they can be individually introduced into a target area or analyzing area. The fluid stream may be induced to form droplets to subsequently aid in the separation of individual particles. Typically, flow cytometers use an electromagnetic radiation emission source such as a laser to generate a beam of electromagnetic radiation that can be directionally controlled to intercept the target area. Irradiation of a target particle passing through the target area, such as a cell, can give rise to scatter or fluorescence emission that can be directionally controlled to a receiver that generates a signal that can be analyzed to differentiate particles.
A significant problem with conventional flow cytometer systems can be that precise analysis of such scatter or fluorescence emission requires that the target area remain precisely aligned with the beam of electromagnetic radiation and that the scatter or fluorescence emission from irradiation of the target remain precisely aligned with the sensor. However, the position of the various components of a flow cytometer change in response to small fluctuations in the external environment, including, but not limited to small fluctuations in temperature, pressure, mechanical forces, as well as small fluctuations in the internal environment, including, but not limited to, electronic drift, radiation frequency or amplitude, or the like. The fluctuations can occur during set up of the instrument or during routine instrument operation raising a variety of concerns.
First, the period of time that an operator expends to align a flow cytometer at the start of an operating period or during routine operation of the flow cytometer can be considerable. It can amount to a significant portion of the operator's scheduled time on the instrument and can abbreviate or even preclude any actual analysis efforts.
Second, monitoring of alignment over long time periods, perhaps hours, can be difficult with the naked eye. Even the smallest variations in alignment can render sensor signals useless or result in increased contamination of sorted particle populations. On-the-fly alignment correction by the flow cytometer operator may not be reliable since the data available to the operator can result in subjective estimates of true alignment.
Third, inconsistency from alignment to alignment can prevent satisfactory calibration of flow cytometers to standardized calibration particles. Often instrument parameters other than alignment are used to calibrate an instrument once the optical alignment is subjectively optimized by an operator. Those familiar with flow cytometry may be aware that instrument calibration performed in this manner can lead to a wide and sometimes unacceptable variation in operating results, even during routine applications.
Fourth, because conventional flow cytometer systems may not have the necessary alignment monitoring equipment to determine if a flow cytometry system is aligned, is slightly out of alignment and requires adjustment, or whether the system is catastrophically mis-aligned, it can be difficult to allow a conventional flow cytometer system to operate unattended by an operator.
Various attempts have been made to hard-mount all optical components in conventional flow cytometer systems to address these concerns. Unfortunately, such flow cytometer systems can still be prone to temperature and mechanical drift and need to be serviced regularly for alignment re-calibration. Moreover, hard mount optical components may only be available with respect to certain cuvette-based flow cytometer systems.
As to the field of flow cytometry and the overall desire to automate and monitor mechanical and optical alignment of flow cytometry systems, the present invention discloses techniques that overcome virtually every one of these problems in a practical fashion.
Perhaps surprisingly, it satisfies a long-felt need to achieve high-speed, accurate, and economical methods for automated positioning of components within the flow cytometer. To some degree, even those involved in the manufacturing of flow cytometers had not appreciated that the problems of monitoring and directionally controlling flow cytometer system alignment could be solved by utilizing the various components disclosed in the present invention.